Tobacco-derived colorants and colored substrates

ABSTRACT

Methods for obtaining natural colorants from plant material (e.g., tobacco material) and for using such natural colorants to dye various substrates are provided. Natural colorants are obtained using particular enzymes and particular conditions (e.g., time, temperature, and pH profiles). Such colorants can be used to dye substrates, for example, using conventional dyeing techniques or using unique in situ methods.

FIELD OF THE INVENTION

The present invention relates to colorants and dyes derived from plants(e.g., tobacco) and to methods for deriving such colorants and dyes. Theinvention further relates to substrates, e.g., fibers and textilestreated with such colorants and dyes and to methods of providing coloredsubstrates.

BACKGROUND OF THE INVENTION

Coloration of various substrates (e.g., textiles) can be achieved usingnatural colorants or synthetic colorants/pigments. Natural colorants,derived, e.g., from plants, fruits, vegetables, insects, and mineralswere among the first colorants and a range of natural colorants areknown and used today for various applications. For example, red colorscan be derived from beets, red cabbage and red radishes; yellow/orangecolors can be derived from annato, beta carotene, saffron, luteines,pomegranate, safflower, and turmeric; green colors can be derived fromchlorophyll; blue/purple colors can be derived from indigo, spirulinaand black carrots; and brown colors can be derived from malt andcaramel. Many natural colorants require the addition of one or morechemicals, e.g., mordants, to ensure binding of the colorant to thesubstrate (particularly for coloring cellulosic fibers). Further, themethods traditionally used for applying natural colorants generallyexhibited poor reproducibility, were time consuming, and were notenvironmentally friendly processes.

There are relatively few known commercial applications of naturalcolorants to textiles, with one principal exception to this observationbeing the application of indigo. Synthetic dyes are thus commonlyemployed for large commercial operations. However, the look of naturaldyes is commonly found to be more aesthetically pleasing. With thegrowth of the biofuel and ethanol industries, knowledge of anddevelopment of aggressive enzymes has grown and these enzymes holdpotential for enhancing the provision and use of certain naturalcolorants. See, e.g., U.S. Pat. No. 8,690,966 and U.S. PatentApplication Publication Nos. 2015/0000050; 2012/0272460; and2011/0287681, all to Devall, which is incorporated herein by reference.It would be beneficial to provide further natural colorants andprocessing techniques that can allow for the production and applicationof such natural colorants to a range of substrates.

SUMMARY OF THE INVENTION

The present disclosure provides natural colorants derived from tobaccomaterials (e.g., tobacco plants or portions thereof). Such colorants canbe used for various purposes, including for dyeing fibers (e.g., naturalfibers). Methods for providing such colorants from tobacco materials arealso provided, which may, in some embodiments, include multipleprocesses (e.g., obtaining the colorants and, optionally, dyeing fibersusing the colorants in a single bath).

The present disclosure provides methods for extracting natural colorantsfrom tobacco plant materials using advanced enzymatic techniques.Specifically, in one aspect, such methods comprise the steps of:providing a first tobacco mixture by combining tobacco material with acellulase and a beta glucosidase at a pH of about 5-6; providing asecond tobacco mixture by adjusting the pH of the first tobacco mixtureto a pH of about 7-8 and adding pectate lyase and xylanase to the firsttobacco mixture; and separating the tobacco material from the secondtobacco mixture to give a colorant-containing broth.

In some embodiments, the tobacco material from which colorants areextracted is a harvested tobacco plant or one or more portions thereof.In some embodiments, the tobacco material is in the form of amanufacturing byproduct or production waste stream.

The first providing step, in some embodiments, comprises combining thetobacco material, cellulase, and beta glucosidase at elevatedtemperature. The elevated temperature can be, for example, at least 38°C., e.g., about 38° C. to about 100° C., about 38° C. to about 75° C. orabout 49° C. to about 71° C. In some embodiments, the first providingstep comprises combining the tobacco material, cellulase, and betaglucosidase with agitation. The first providing step, in certainembodiments, comprises adding a pH modifier (e.g., a buffer) to obtainthe pH of about 5-6. Exemplary pH modifiers include, but are not limitedto, monosodium phosphate, disodium phosphate, and combinations thereof.In the first providing step, the cellulase and beta glucosidase are, insome embodiments, in a ratio of about 10:1 to about 3:1. In certainembodiments, the first providing step comprises preparing an enzymemixture comprising the cellulase and beta glucosidase, adjusting the pHof the enzyme mixture, and combining the enzyme mixture and the tobaccomaterial.

The second providing step, in some embodiments, comprises combining thefirst tobacco mixture, the pectate lyase, and the xylanase at elevatedtemperature. The elevated temperature can be, for example, at least 38°C., e.g., about 38° C. to about 100° C., about 38° C. to about 75° C.,or about 49° C. to about 71° C. In some embodiments, the secondproviding step comprises combining the first tobacco mixture, pectatelyase, and xylanase with agitation. The second providing step, incertain embodiments, comprises adding a pH modifier (e.g., a buffer) toobtain a pH of about 7-8. Exemplary pH modifiers include, but are notlimited to, monosodium phosphate, disodium phosphate, and combinationsthereof. In the second providing step, the xylanase and pectate lyaseare, in some embodiments, in a ratio of about 1:1 to about 5:1. Incertain embodiments, the second providing step comprises preparing anenzyme mixture comprising the xylanase and pectate lyase, adjusting thepH of the enzyme mixture, and combining the enzyme mixture and the firsttobacco mixture.

The colorants generated according to the disclosed methods can be usedas coloring agents for a wide variety of materials/substrates. Forexample, such colorants can be used as inks, e.g., to color papers,plastics, ceramics, metals, and other types of materials and asdyes/pigments for textiles, e.g., to color fabrics such as for clothing,homegoods (e.g., linens, tablecloths, curtains, rugs, towels, andupholstered furnishings), automobile components, and the like. Thedisclosed colorants can also find use in tobacco products, including,but not limited to, as inks for product packaging and/or cigarettepaper; as dyes for filter tow material and snus pouches; and ascomponents of e-cigarettes. Furthermore, the colorants may be useful inother types of products, e.g., as food dyes, cosmetic colorants, and inpaint formulations.

The present disclosure further provides methods for dyeing varioussubstrates with natural colorants extracted from tobacco plantmaterials. The dyeing can, in some embodiments, be done subsequent tothe provision of the colorant-containing broth by contacting a substratewith the colorant-containing broth. The dyeing can, in some embodiments,can be done in tandem with the provision of the colorant-containingbroth (e.g., wherein the substrate is added during the extractionprocess). Advantageously, in certain embodiments, the dyeing can beconducted “in situ” within the same bath as the extracting by adding asubstrate at any stage during or following the extracting methodperformed to provide the colorant-containing broth.

As such, in one aspect, a method of dyeing a substrate is provided,comprising: extracting colorant from a tobacco material according to themethods disclosed herein and contacting a substrate with the colorant.This method can comprise performing the extracting method and thenadding a substrate. The method can comprise adding the substrate duringthe extracting method (e.g., during the first providing step or duringthe second providing step). The present disclosure also provides dyedsubstrates exhibiting certain desirable features, as will be describedin further detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to provide an understanding of embodiments of the invention,reference is made to the appended drawings, which are not necessarilydrawn to scale, and in which reference numerals refer to components ofexemplary embodiments of the invention. The drawings are exemplary only,and should not be construed as limiting the invention.

FIG. 1 is a schematic diagram providing steps of an exemplary method forthe provision of tobacco-derived colorant according to one embodiment;

FIG. 2 is a schematic diagram providing steps of an exemplary method forthe provision of tobacco-derived colorant according to a furtherembodiment;

FIG. 3A is a L*a*b* plot of a cotton substrate dyed with tobacco-derivedcolorant; FIG. 3B is a reflectance plot of the same dyed substrate; and

FIG. 4A is a L*a*b* plot of a cotton substrate dyed with tobacco-derivedcolorant; FIG. 4B is a reflectance plot of the same dyed substrate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. As used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Reference to “dry weight percent” or“dry weight basis” refers to weight on the basis of dry ingredients(i.e., all ingredients except water).

In a first aspect, the present disclosure provides a method forobtaining natural colorants and/or dyes from plant material (e.g.,tobacco material). This method generally involves extracting a coloredcomponent from the tobacco material and, in particular, extracting acolored component using particular enzymes and particular conditions(e.g., time, temperature, and pH profiles). Advantageously, in someembodiments, such methods can maximize the amount of colorant releasedfrom the tobacco material and/or can minimize the biomass remainingafter the extraction process.

One exemplary method for obtaining natural colorant from a tobaccomaterial is set forth in the schematic diagram of FIG. 1, which isdescribed as follows. Tobacco Material 120 is combined with First EnzymeMixture 110 to give Tobacco Mixture 1 130 and treated 20 to give TreatedTobacco Mixture 1 140. Treated Tobacco Mixture 1 140 is combined withSecond Enzyme Mixture 150 to give Tobacco Mixture 2 160 and treated 22to give the desired colorant 170. One embodiment of such a method isfurther outlined in the schematic diagram of FIG. 1, in which Cellulase112, Beta-glucosidase 114, and pH modifier 116 are separately or in anycombination combined with Tobacco Material 120 to give Tobacco Mixture 1130, which is treated 20 to give Treated Tobacco Mixture 1 140.Subsequently, pectate lyase 152, xylanase 154, and pH modifier 156 areseparately or in any combination combined with Treated Tobacco Mixture 1140 to give Tobacco Mixture 2 160, which is treated 22 to give thecolorant 170.

It is noted that certain processing parameters with respect to themethod steps (e.g., time, temperature, pH profile, specific enzyme(s),order of addition of enzymes) identified by the inventors have led, insome cases, to significantly improved efficacy of colorant extractionfrom tobacco, as will be detailed further herein. One of skill in theart will recognize that FIGS. 1 and 2 provide only general overviews ofparticular embodiments of the method of the invention and willappreciate that the referenced components in this process can includeadditional components and that additional method steps may be includedin the process.

The “tobacco material” 120 subjected to the methods disclosed hereinencompasses a range of tobacco-containing materials. For example, insome embodiments, the tobacco material comprises tobacco plants orportions thereof, in various stages of the plant life cycle. Suchtobacco plants or portions thereof include, but are not limited to,uncured tobacco plants or portions, i.e., plants or portions thereof in“green” form. By “green” in certain embodiments is meant tobacco havinga form such that cells within the plant or plant component have notexperienced significant or substantial cell death, and cellularrespiration is capable of occurring to some degree. Such tobacco plantsor portions thereof in “green” form can be processed according to thedisclosed methods at various time points post-harvest (e.g., immediatelyafter harvest or at some stage thereafter). In certain embodiments, thetobacco plants or portions thereof are processed prior to being dried(i.e., shortly after harvest).

In other embodiments, the tobacco material comprises tobacco that hasbeen cured and/or aged. Methods for curing and aging are generally knownand examples of methods for curing and/or aging tobacco are discussed,for example, in U.S. Pat. No. 1,327,692 to Beinhart; U.S. Pat. No.2,758,603 to Heljo; U.S. Pat. No. 5,676,164 to Martin; U.S. Pat. No.6,755,200 to Hempfling et al.; U.S. Pat. No. 7,293,564 to Perfetti etal., and U.S. Pat. No. 8,353,300 to Li et al.; and US Pat. Appl. Pub.Nos. 2010/0116281 and 2012/0279510 to Marshall et al., which areincorporated herein by reference in their entireties. Descriptions offurther types of curing and aging processes for various types of tobaccoare provided in Tobacco Production, Chemistry and Technology, Davis etal. (Eds.) (1999), which is also incorporated herein by reference.

For example, tobacco can be cured by methods including but not limitedto, air-curing, dark air curing, sun-curing, fire curing, and fluecuring. Flue curing comprises curing tobacco in enclosures wherein fluesheat cure the tobacco without exposing it to smoke and is described, forexample, in Nestor et al. Beitrage Tabakforsch. Int., 2003, 20, 467-475and U.S. Pat. No. 6,895,974 to Peele, which are both incorporated hereinby reference. Fire cured tobacco generally comprises curing tobacco inenclosures wherein it is exposed to the gaseous combustion products of afire that is maintained at a low smolder and is described, for example,in US Pat. Appl. Publ. 2012/0125354 to Byrd et al., which isincorporated herein by reference. Air curing typically comprises hangingtobacco in a well-ventilated enclosure to dry at ambient conditions andis described, for example, in deRoton, C. et al. Beitrage Tabakforsch.Int. 2005, 21, 6, 305-320; Staaf, M. et al. Beitrage Tabakforsch Int.2005, 21, 6, 321-330; and U.S. Pat. No. 6,834,654 to Williams, which areincorporated herein by reference. Sun curing generally comprisesallowing tobacco to cure uncovered in the sun.

Tobacco material 120 can comprise varying portions of a tobacco plant.For example, virtually all of the plant (e.g., the whole plant) can beharvested and employed as such. Alternatively, various parts or piecesof the plant can be harvested or separated for treatment after harvest.For example, the flower, leaves, stem, stalk, roots, seeds, and variouscombinations thereof, can be isolated for use or further treatment.Tobacco is commonly grown for its leaves, which are harvested and used,e.g., for smoking products and smokeless tobacco products. Otherportions of the tobacco plant (e.g., the flower, stem, stalk, roots, andseeds) are usually less widely used (and sometimes discarded). Incertain embodiments, such portions can be advantageously processedaccording to the disclosed methods. In particular embodiments, thetobacco material subjected to processing as disclosed herein is tobaccomaterial other than tobacco leaf, including the portions referencedherein above as well as tobacco material that is generally considered tobe “waste” or scrap (e.g., tobacco material remaining after variousmanufacturing or other processes).

In some embodiments, tobacco material 120 subjected to methods asdisclosed herein can comprise a mixture of components, e.g., in the formof a manufacturing byproduct or production waste stream. As such, the“tobacco material” can be provided in a mixture with one or more othercomponents, e.g., including but not limited to such compounds as sugars,proteins, undigested tobacco material, and tobacco-derived compounds. Assuch, the method described herein may, in some embodiments, be employedin the context of recycling waste material. Byproducts or productionwaste streams can be treated prior to processing as disclosed herein,e.g., to concentrate or remove certain components therefrom or can beused directly.

Tobacco material 120 can be used in intact form (e.g., in the form ofplants or plant parts) or can be physically processed prior to beingsubjected to the disclosed method. For example, a tobacco plant orportion thereof can be separated into individual parts or pieces (e.g.,leaves can be removed from stems, and/or stems and leaves can be removedfrom stalk). A plant or individual parts or pieces can be furthersubdivided into parts or pieces (e.g., leaves can be shredded, cut,comminuted, pulverized, milled or ground into pieces or parts that canbe characterized as filler-type pieces, granules, particulates or finepowders). The tobacco material can, in some embodiments, be extractedprior to processing according to the methods disclosed herein (whereeither the extracted tobacco pulp or the tobacco extract can serve astobacco material 120). Exemplary methods for extracting variouscomponents from tobacco include, but are not limited to, those describedin U.S. Patent Application Publication No. 2011/0247640 to Beeson et al.and U.S. Pat. No. 4,144,895 to Fiore; U.S. Pat. No. 4,150,677 toOsborne, Jr. et al.; U.S. Pat. No. 4,289,147 to Wildman et al.; U.S.Pat. No. 4,359,059 to Brummer et al.; U.S. Pat. No. 4,589,428 toKeritsis; U.S. Pat. No. 4,605,016 to Soga et al.; U.S. Pat. No.4,727,889 to Niven, Jr. et al.; U.S. Pat. No. 4,941,484 to Clapp et al.;U.S. Pat. No. 4,967,771 to Fagg et al.; U.S. Pat. No. 4,986,286 toRoberts et al.; U.S. Pat. No. 5,121,757 to White et al.; U.S. Pat. No.5,131,414 to Fagg; U.S. Pat. No. 5,131,415 to Munoz et al.; U.S. Pat.No. 5,148,819 to Fagg; U.S. Pat. No. 5,435,325 to Clapp et al.; U.S.Pat. No. 5,445,169 to Brinkley et al.; U.S. Pat. No. 6,131,584 toLauterbach; U.S. Pat. No. 6,298,859 to Kierulff et al.; U.S. Pat. No.6,772,767 to Mua et al.; and U.S. Pat. No. 7,337,782 to Thompson, whichare all incorporated herein by reference.

Where some or all of tobacco material 120 comprises roots and/or stalks,such components are, in some embodiments, advantageously cleaned and/orphysically processed prior to use. In some embodiments, certaincomponents, e.g., pith, parenchyma, and other non-woody materials areremoved prior to treatment as disclosed herein. Roots and stalks inparticular may, in some embodiments, be processed according to themethods set forth in U.S. Patent Application Publication Nos.2012/0152265 and 2012/0192880, both to Dube et al., which areincorporated herein by reference. In certain embodiments, thesecomponents can be cleaned and processed using methods and equipmentgenerally employed for green leaf processing. One particular method forcleaning and processing stalks includes the steps of: size reduction(e.g., by milling and threshing), screening (to give a materialcollected on the screen comprising cleaned root/stalk product and ascreened fine material containing pith, parenchyma, and small woodypieces); and passing the small woody pieces through an air separator(which separates the pieces by density, removing non-woody pieces suchas pith and parenchyma). One particular method for cleaning andprocessing roots includes the steps of size reduction (e.g., bymilling); screening to remove dirt and fines; air separation to removelight non-woody material; threshing; additional air separation to removelight non-woody material; air separation to remove heavy contaminants;manual picking to remove foreign matter; screening (to give a materialcollected on the screen comprising cleaned root and a screened materialwhich can be further used); and air separation of both materials toremove light non-woody material.

Tobacco or tobaccos to which the methods provided herein are applicablecan vary. In certain embodiments, tobaccos that can be employed includeflue-cured or Virginia (e.g., K326), burley, sun-cured (e.g., IndianKurnool and Oriental tobaccos, including Katerini, Prelip, Komotini,Xanthi and Yambol tobaccos), Maryland, dark, dark-fired, dark air cured(e.g., Pasado, Cubano, Jatim and Bezuki tobaccos), light air cured(e.g., North Wisconsin and Galpao tobaccos), Indian air cured, RedRussian and Rustica tobaccos, as well as various other rare or specialtytobaccos and various blends of any of the foregoing tobaccos.Descriptions of various types of tobaccos, growing practices andharvesting practices are set forth in Tobacco Production, Chemistry andTechnology, Davis et al. (Eds.) (1999), which is incorporated herein byreference. Various representative other types of plants from theNicotiana species are set forth in Goodspeed, The Genus Nicotiana,(Chonica Botanica) (1954); U.S. Pat. No. 4,660,577 to Sensabaugh, Jr. etal.; U.S. Pat. No. 5,387,416 to White et al. and U.S. Pat. No. 7,025,066to Lawson et al.; US Patent Appl. Pub. Nos. 2006/0037623 to Lawrence,Jr. and 2008/0245377 to Marshall et al.; each of which is incorporatedherein by reference. Exemplary Nicotiana species include N. tabacum, N.rustica, N. alata, N. arentsii, N. excelsior, N. forgetiana, N. glauca,N. glutinosa, N. gossei, N. kawakamii, N. knightiana, N. langsdorffi, N.otophora, N. setchelli, N. sylvestris, N. tomentosa, N. tomentosiformis,N. undulata, N. x sanderae, N. africana, N. amplexicaulis, N.benavidesii, N. bonariensis, N. debneyi, N. longiflora, N. maritina, N.megalosiphon, N. occidentalis, N. paniculata, N. plumbaginifolia, N.raimondii, N. rosulata, N. simulans, N. stocktonii, N. suaveolens, N.umbratica, N. velutina, N. wigandioides, N. acaulis, N. acuminata, N.attenuata, N. benthamiana, N. cavicola, N. clevelandii, N. cordifolia,N. corymbosa, N. fragrans, N. goodspeedii, N. linearis, N. miersii, N.nudicaulis, N. obtusifolia, N. occidentalis subsp. Hersperis, N.pauciflora, N. petunioides, N. quadrivalvis, N. repanda, N.rotundifolia, N. solanifolia, and N. spegazzinii.

Nicotiana species can be derived using genetic-modification orcrossbreeding techniques (e.g., tobacco plants can be geneticallyengineered or crossbred to increase or decrease production ofcomponents, characteristics or attributes). See, for example, the typesof genetic modifications of plants set forth in U.S. Pat. No. 5,539,093to Fitzmaurice et al.; U.S. Pat. No. 5,668,295 to Wahab et al.; U.S.Pat. No. 5,705,624 to Fitzmaurice et al.; U.S. Pat. No. 5,844,119 toWeigl; U.S. Pat. No. 6,730,832 to Dominguez et al.; U.S. Pat. No.7,173,170 to Liu et al.; U.S. Pat. No. 7,208,659 to Colliver et al. andU.S. Pat. No. 7,230,160 to Benning et al.; US Patent Appl. Pub. No.2006/0236434 to Conkling et al.; and PCT WO 2008/103935 to Nielsen etal. See, also, the types of tobaccos that are set forth in U.S. Pat. No.4,660,577 to Sensabaugh, Jr. et al.; U.S. Pat. No. 5,387,416 to White etal.; and U.S. Pat. No. 6,730,832 to Dominguez et al., each of which isincorporated herein by reference. Most preferably, the tobacco materialsare those that have been appropriately cured and aged. Especiallypreferred techniques and conditions for curing flue-cured tobacco areset forth in Nestor et al., Beitrage Tabakforsch. Int., 20 (2003)467-475 and U.S. Pat. No. 6,895,974 to Peele, which are incorporatedherein by reference. Representative techniques and conditions for aircuring tobacco are set forth in deRoton, C. et al. Beitrage Tabakforsch.Int., 2005, 21, 6, 305-320 and Staaf, M. et al. Beitrage Tabakforsch.Int. 2005, 21, 6, 321-330, which are incorporated herein by reference.Certain types of unusual or rare tobaccos can be sun cured.Representative Oriental tobaccos include katerini, prelip, komotini,xanthi and yambol tobaccos. Tobacco compositions including dark aircured tobacco are set forth in US Patent Appl. Pub. No. 2008/0245377 toMarshall et al., which is incorporated herein by reference. See also,types of tobacco as set forth, for example, in US Patent Appl. Pub. No.2011/0247640 to Beeson et al., which is incorporated herein byreference. The Nicotiana species can be selected for the content ofvarious compounds that are present therein. Tobacco plants can be grownin greenhouses, growth chambers, or outdoors in fields, or grownhydroponically.

Tobacco material 120 can optionally be physically processed prior tobeing subjected to the methods disclosed herein. The plant or portionthereof can be separated into individual parts or pieces (e.g., theleaves can be removed from the stems, the stems and leaves can beremoved from the stalk, stalk can be removed from the roots, etc.). Theharvested plant or individual parts or pieces can be further subdividedinto parts or pieces (e.g., the leaves can be shredded, cut, comminuted,pulverized, milled or ground into pieces or parts that can becharacterized as pieces, granules, particulates or fine powders). Thetobacco material can have the form of processed tobacco parts or pieces,cured and aged tobacco in essentially natural lamina, stem, stalk, orroot form, a tobacco extract, extracted tobacco pulp (e.g., using wateras a solvent), or a mixture of the foregoing.

The manner by which the tobacco is provided in such forms can vary. Theplant, or parts thereof, can be subjected to external forces or pressure(e.g., by being pressed or subjected to roll treatment). When carryingout such processing conditions, the plant or portion thereof can have amoisture content that approximates its natural moisture content (e.g.,its moisture content immediately upon harvest), a moisture contentachieved by adding moisture to the plant or portion thereof, or amoisture content that results from the drying of the plant or portionthereof. For example, powdered, pulverized, ground or milled pieces ofplants or portions thereof can have moisture contents of less than about25 weight percent, often less than about 20 weight percent, andfrequently less than about 15 weight percent. Tobacco parts or piecescan be comminuted, ground or pulverized into a powder type of form usingequipment and techniques for grinding, milling, or the like. Mostpreferably, the tobacco is relatively dry in form during grinding ormilling, using equipment such as hammer mills, cutter heads, air controlmills, or the like. In some embodiments, the specific features of aprocessed tobacco material can affect the resulting color and hue of thecolorant-containing broth/dye produced therefrom (e.g., the level offineness of the grind).

The composition of the first enzyme mixture 110 of FIG. 1 can vary.Generally, this first enzyme mixture is aqueous and includes at leastone enzyme. In certain preferred embodiments, the first enzyme mixturecomprises a cellulase and a glucosidase. As further represented in FIG.2, cellulase 112 and glucosidase 114 can be provided either in the formof a mixture (110 of FIG. 1) that can be combined with Tobacco Material120 or can be separately combined therewith to give Tobacco Mixture 1130. As such, the components employed to produce Tobacco Mixture 1 130can be added in various sequences. For example, water and pH modifier116 can be combined and mixed with the tobacco material, followed by theaddition of the cellulase 112 and beta-glucosidase 114. As anotherexample, water and pH modifier 116 can be combined and the cellulase andbeta-glucosidase can be added, followed by the addition of the tobaccomaterial. As such, referring back to FIG. 1, although first enzymemixture 110 is referred to generally as a “mixture,” in variousembodiments, the components can be considered to be “mixed” before,during, or after contact with Tobacco Material 120 to provide TobaccoMixture 1 130. In certain embodiments, one or more of the components maybe provided in a preformed mixture. For example, a commercial enzymemixture comprising one or more cellulases and one or more glucosidasesmay be used. One such commercially available mixture from DuPont™Enzymes is MULTIFECT® CX.

Cellulases are a family of enzymes capable of breaking down(hydrolyzing) the cellulose in plant material. Various structurally andmechanistically different cellulases are known, and include, but are notlimited to, endocellulases, exocellulases, cellobiases, oxidativecellulases, and cellulose phosphorylases. Certain specific cellulasesinclude endo-1,4-beta-D-glucanase, carboxymethyl cellulase, avicelase,celludextriase, cellulase A, cellulosin AP, alkali cellulase, cellulaseA 3, 9.5 cellulase, and pancellase SS. Such enzymes are commerciallyavailable, e.g., from DuPont™ Enzymes (including PRIMAFAST® familycellulases, including GOLD HSL, LUNA, LMA 400, and 200; OPTIMASE® familycellulases, including OPTIMASE CX 57E; and INDIAGE® family cellulases,including NeutraFlex, Super L, and Super GX Plus), AB Enzymes (BIOTOUCH®family cellulases and ECOSTONE® family cellulases). Any single cellulaseor combination of cellulases can be employed as the cellulase componentherein. The concentration of cellulase in the Tobacco Mixture 1 130 canbe any amount sufficient to provide some degree of hydrolysis of thecellulose in the tobacco material. Exemplary concentrations of the oneor more cellulases present therein include, but are not limited to,about 30 to about 50 weight percent of the enzymes and water present inTobacco Mixture 1 130.

Glucosidases are a class of enzymes involved in breaking down glycogensinto glucose. Exemplary glucosidases include, but are not limited to,α-glucosidase and β-glucosidase. Although any glucosidase can beemployed as the glucosidase component of the first enzyme mixture, theglucosidase in some embodiments is advantageously beta-glucosidase.Beta-glucosidase specifically functions by hydrolyzing terminal,non-reducing β-D-glucosyl residues, releasing β-D-glucose. Theconcentration of glucosidase included in Tobacco Mixture 1 130 can beany amount sufficient to provide some degree of breakdown of theglycogens of the tobacco material. Exemplary concentrations of the oneor more glucosidases present therein include, but are not limited to,about 0 to about 50 weight percent, e.g., about 5 to about 50 weightpercent or about 15 to about 35 weight percent of the enzymes and waterpresent in Tobacco Mixture 1 130.

A pH modifier is generally also employed in combination with thecellulase and beta-glucosidase to treat the Tobacco Material 120 and, asreferenced above, the pH modifier 116 can be added in combination withone or more of the enzymes (e.g., as part of First Enzyme Mixture 110)or can be independently added. Advantageously, the pH of Tobacco Mixture1 130 is about 5 to about 6.5, e.g., about 5.5 to about 6. pH modifiersinclude acids and bases and, particularly include buffers (i.e., certaincombinations of acids and bases that are used to maintain a relativelyconstant pH). Compounds capable of modifying the pH can vary. Generally,any compounds known to increase or decrease the pH of a solution and/orknown to buffer a solution around a given pH (depending on the startingmaterial of the solution and the target final pH) can be used for thispurpose. Representative compounds capable of modifying the pH include,but are not limited to, phosphates, acetates, citrates, carbonates, andsulfonates, with certain specific examples including, but not limitedto, sodium phosphates (e.g., monosodium phosphate (MSP)) disodiumphosphate (DSP), trisodium phosphate, monosodium diphosphate, disodiumdiphosphate, trisodium diphosphate, and tetrasodium diphosphate); sodaash, and acetic acid.

Exemplary amounts of such compounds incorporated within Tobacco Mixture1 130 will vary depending on the starting pH of the mixture, the targetpH, and the particular pH modifier(s) used. In certain embodiments, theamount of pH modifier included is that amount to provide a pH of about5.5-6.0 and/or to buffer within this pH range. For example, when the pHmodifier is MSP, a representative amount in Tobacco Mixture 1 130 may beabout 5-15 g/L or about 7 to about 12 g/L, e.g., about 10 g/L. It isnoted that certain enzymes operate only within certain pH ranges;accordingly, the pH in some embodiments must be adjusted to ensure goodactivity of the enzyme(s) in Tobacco Mixture 1 130.

The relative amounts of enzymes, pH modifier, water, and tobaccomaterial in Tobacco Mixture 1 130 can vary. Typically, the combined dryweight of enzymes in this particular step is somewhat less than the dryweight of the tobacco material, but the amount of tobacco material inTobacco Mixture 1 130 is not particularly limited. Although not intendedto be limiting, in some embodiments, the total enzyme amount (by weight)with respect to the dry tobacco material in Tobacco Mixture 1 is roughly50%. For example, in some embodiments, the total enzyme amount (byweight) is about 25% the dry weight of the tobacco material to about 75%the dry weight of the tobacco material. The ratio of cellulase tobeta-glucosidase is generally such that a greater amount by weight ofcellulase is present as compared to the amount of beta-glucosidase. Forexample, the cellulase and beta-glucosidase can be in a weight ratio ofabout 10:1 to about 3:1.

The conditions of step 20, in which Tobacco Mixture 1 130 is treated canvary. For example, this mixture can be treated at various temperatures,under various atmospheric conditions, and for various periods of time.Desirably, in some embodiments, application of heat can promote enzymeactivity and as such, step 20 can, in some embodiments, compriseapplying heat to Tobacco Mixture 1 130; however, useful temperaturesdepend on the particular enzyme(s) present in the enzyme mixture. Incertain embodiments, the components can be combined and heated at atemperature of at least about 100° F. (˜38° C.), at least about 120° F.(˜49° C.), or at least about 140° F. (60° C.). Exemplary temperatureranges include about 100° F. to about 200° F. (˜38° C. to ˜93° C.) andabout 120° F. to about 160° F. (˜49° C. to ˜71° C.). Exemplary timeranges for which the enzyme mixture and tobacco material are kept incontact under such conditions are not particularly limited and includeat least about 1 hour, at least about 2 hours, at least about 3 hours,or at least about 4 hours, e.g., about 1-24 hours, 2-10 hours, or 4-6hours. The components can be combined with or without agitation,although agitation (e.g., constant agitation) may be desirable topromote enzyme activity.

The resulting, treated tobacco mixture, Treated Tobacco Mixture 1 140 ofFIG. 1 is further combined with a second aqueous enzyme mixture 150,which in preferred embodiments includes a pectate lyase and a xylanase.As further represented in FIG. 2, pectate lyase 152 and xylanase 154 canbe provided either in the form of a mixture (150 of FIG. 1) that can becombined with Treated Tobacco Mixture 1 140 or can be separatelycombined therewith. As such, the components employed to produce TobaccoMixture 2 160 can be added in various sequences. For example, pHmodifier 156 as shown in FIG. 2 can be added to Treated Tobacco Mixture1, followed by the addition of the enzymes, which may be addedseparately or together. in some embodiments, additional water is addedto Treated Tobacco Mixture 1, either separately or in combination withone or more of pH modifier 156, xylanase 154, or pectate lyase 152.Referring back to FIG. 1, although Second Enzyme Mixture 150 is referredto generally as a “mixture,” in various embodiments, the components canbe considered to be “mixed” before, during, or after contact withTreated Tobacco Mixture 1 140 to provide Tobacco Mixture 2 160.

Pectate lyases, also referred to as pectate transeliminases, are enzymesexhibiting pectate lyase activity, i.e., enzymes that are responsiblefor the eliminative cleavage of de-esterified pectin, generatingoligosaccharides with unsaturated galacturonosyl residues at theirnon-reducing ends. Various pectate lyases are described, for example, inU.S. Pat. No. 6,124,127 to Andersen et al., which is incorporated hereinby reference. Commercial pectate lyases are available, etc. from DuPont™Enzymes (including PRIMAGREEN® family pectate lyases, such asPrimaGreen® EcoScour). The concentration of cellulase in Tobacco Mixture2 160 can vary and can be any amount sufficient to provide some degreeof cleavage of de-esterified pectin. Exemplary concentrations of thepectate lyase(s) present in Tobacco Mixture 2 160 include, but are notlimited to, about 0 to about 50 weight percent, e.g., about 5 to about50 weight percent or about 15 to about 35 weight percent of the enzymesand water present in Tobacco Mixture 2 160. The amount of pectate lyaseused with respect to the dry tobacco weight can also vary, e.g., fromabout 5% by weight with respect to the tobacco weight to about 100% byweight of the dry tobacco weight.

Xylanases are hydrolytic enzymes which randomly cleave thebeta-1,4-backbone of xylan (a polysaccharide found in plant cell walls).Xylanases can range in the numbers/types of folds, specific mechanismsof action, substrate specificities, yields, rates, products, andphysicochemical characteristics. Six xylanase-containing enzyme familiesare known and can be employed according to the present methods.Commercial xylanases are available, etc. from DuPont™ Enzymes (includingOPTIMASE® family enzymes, such as OPTIMASE CX 72L and CX 255L).Exemplary concentrations of the one or more xylanases present in TobaccoMixture 2 160 include, but are not limited to, about 0 to about 50weight percent, e.g., about 5 to about 50 weight percent or about 15 toabout 35 weight percent of the enzymes and water present in TobaccoMixture 2 160. The amount of pectate lyase used with respect to the drytobacco weight can also vary, e.g., from about 5% by weight with respectto the tobacco weight to about 100% by weight of the dry tobacco weight.[

A pH modifier is generally also employed in combination with the pectatelyase and xylanase to treat Treated Tobacco mixture 1 and, as referencedabove, the pH modifier 156 can be added in combination with one or moreof the enzymes (e.g., as part of second Enzyme Mixture 150) or can beindependently added. Advantageously, the pH of Tobacco Mixture 2 isabout 7.5-8. Compounds capable of modifying the pH can vary. Generally,any compounds known to increase or decrease the pH of a solution or tobuffer a solution around a given pH (depending, e.g., on the starting pHof Treated Tobacco Mixture 1 140, and the target pH for Tobacco Mixture2 160) can be used for this purpose. Representative compounds capable ofmodifying the pH include, but are not limited to, phosphates, acetates,citrates, carbonates, and sulfonates, with certain specific examplesincluding, but not limited to, sodium phosphates (e.g., monosodiumphosphate (MSP)) disodium phosphate (DSP), trisodium phosphate,monosodium diphosphate, disodium diphosphate, trisodium diphosphate, andtetrasodium diphosphate); soda ash, and acetic acid. Exemplary amountsof such compounds incorporated within Tobacco Mixture 2 160 will varydepending on the pH of the mixture without such compounds added, thetarget pH, and the particular compound used. In certain embodiments, theamount of pH modifier included is that amount to provide a pH of about7.5-8. For example, when the pH modifier is DSP, a representative amountmay be about 10 to about 30 g/L or about 15 to about 25 g/L, e.g., about20 g/L. It is noted that certain enzymes operate only within certain pHranges; accordingly, the pH in some embodiments must be adjusted toensure good activity of the enzymes in Tobacco mixture 2 160. Oneparticular Tobacco Mixture 2 160 comprises disodium phosphate (DSP),pectase lyase, xylanase, and water.

The relative amounts of enzymes, pH modifier, water, and tobaccomaterial in Tobacco mixture 2 160 can vary. Typically, the combined dryweight of enzymes in this particular step is somewhat less than the dryweight of the tobacco material, but the amount of tobacco material inTobacco Mixture 2 160 is not particularly limited. Although not intendedto be limiting, in some embodiments, the total enzyme amount (by weight)with respect to the dry tobacco material in Tobacco Mixture 2 is roughly50%. For example, in some embodiments, the total enzyme amount (byweight) is about 25% the dry weight of the tobacco material to about 75%the dry weight of the tobacco material. The ratio of xylanase to pectatelyase is generally such that a greater amount of xylanase with respectto pectate lyase is provided. For example, the xylanase and pectatelyase can, in some embodiments, be present in a weight ratio of about1:1 to about 5:1.

The conditions of step 22, in which second enzyme mixture 140 iscombined with Tobacco Mixture 1 130 can vary. For example, thecomponents can be combined at various temperatures, under variousatmospheric conditions, and for various periods of time. Desirably, insome embodiments, application of heat can promote enzyme activity;however, useful temperatures depend on the particular enzyme(s) presentin the enzyme mixture. In certain embodiments, the components can becombined and heated at a temperature of at least about 100° F. (˜38°C.), at least about 120° F. (˜49° C.), or at least about 140° F. (60°C.). Exemplary temperature ranges include about 100° F. to about 200° F.(˜38° C. to ˜93° C.) and about 120° F. to about 160° F. (˜49° C. to ˜71°C.). Exemplary time ranges for which the enzyme mixture and tobaccomaterial are kept in contact under such conditions are not particularlylimited and include at least about 1 hour, at least about 2 hours, atleast about 3 hours, at least about 4 hours, or at least about 6 hourse.g., about 1-10 hours, 2-8 hours, or 6-8 hours. The components can becombined with or without agitation, although agitation (e.g., constantagitation) may be desirable to promote enzyme activity.

In particular embodiments, step 22 comprises a multiple-stage (e.g.,two-stage) heating process, wherein the combined material is heated at afirst temperature (e.g., about 60-70° C.) for a first period of time(e.g., at least about 4 hours or at least about 6 hours, e.g., 6-8hours), and then the temperature is increased to a second, highertemperature (e.g., about 80-90° C.) for a second period of time (e.g.,at least about 10 minutes, e.g., about 10 minutes to about an hour,e.g., 20-40 minutes). Advantageously, in such multiple-stage heatingprocesses, both stages are conducted with agitation, although agitationis not required in either step.

Following contact between Tobacco Mixture 1 130 and Second EnzymeMixture 140 and advantageously, heating in Step 22 as outlined hereinabove, the mixture is cooled. Methods of cooling and the atmosphereunder which the mixture is cooled can vary. In one particularembodiment, the mixture is cooled to room temperature via radiation orvia cooling coils; however, any means by which cooling can be effectedcan be employed (including simply allowing the mixture to cool withoutany means to promote cooling). The mixture is typically then filtered orotherwise processed to remove any solid material present therein (e.g.,by passing the mixture through a coarse strainer) to give acolorant-containing “broth.” The broth can be used directly as a dye orcan be processed in some manner prior to use. For example, the broth canbe diluted or concentrated by the addition or removal of liquidtherefrom, as desired. in some embodiments, one or more additionalcomponents can be added to the broth, e.g., an additive to intensify thecolor, produce a different shade/hue, or to stabilize the dye.

The selection of enzymes and the sequence of enzyme addition (e.g.,time, temperature and pH profiles) have been found to produce a naturalcolorant-containing broth that is superior to that produced viapreviously reported methods. The methods generally disclosed herein, forexample, lead to enhanced degradation of the cellulosic structure of thetobacco plant as compared with previous methods, providing more colorantand less bio-mass remaining upon completion of the colorant extractionprocess. The methods disclosed herein provide an improved gamut ofcolors in the colorants and such methods are demonstrated to be robustand readily reproducible.

In one particular embodiment, the method for providing a tobacco-derivedcolorant specifically comprises: a) combining a tobacco material, water,a pH modifier, cellulase, and beta glucosidase to give a first mixturehaving a pH of about 5-6; b) heating the first mixture; c) adding asecond pH modifier, pectate lyase, and xylanase to the heated firstmixture to give a second mixture having a pH of about 7-8; d) heatingthe second mixture; e) cooling the heated second mixture; and f)removing solid tobacco material from the cooled mixture to give acolorant-containing broth.

The broths can contain various components in addition to the one or morechromophores that provide the color. In some embodiments, colorantbroths provided herein can contain a significant amount of one or moresugars (e.g., including but not limited to, glucose, fructose, andsucrose) and sugar derivatives. In some embodiments, the colorants cancomprise between about 5 and about 25% by dry weight of one or moresugars and sugar derivatives. Advantageously, the broths containrelatively low amounts of alkaloids, e.g., in certain embodiments, thebroths contain less than about 5% alkaloids by dry weight or less thanabout 3% alkaloids by dry weight.

Furthermore, the methods for colorant extraction outlined herein havebeen found to provide broths comprising tobacco-derived colorants withunique spectral profiles. Such spectral profiles are believed to be verydifficult, and likely impossible to reproduce, using synthetic dyes.Exemplary L*a*b* plots (FIGS. 3A and 4A) and ultraviolet/visible(UV/Vis) spectra (FIGS. 3B and 4B) for substrates dyed withtobacco-derived colorants provided according to the methods outlinedherein are provided. As observed, the UV/Vis spectra of fabrics dyedwith tobacco-derived colorants exhibit a unique, straight line profile.

The natural colorants extracted from plant materials as detailed hereinabove can be employed in various specific methods to endow varioussubstrates with color. As such, one aspect of the present disclosureprovides methods for dyeing substrates with colorants derived fromplants of the Nicotiana species. Such methods can, in some embodiments,be analogous to methods generally used to color substrates includingfibers, textiles and fabrics. Advantageously, the colorants disclosedherein do not require the inclusion of a mordant (e.g., a toxic, heavymetal mordant) in the dye solution to ensure binding of the colorant tothe substrate. It is noted, however, that in some embodiments, use of amordant can be beneficial for maximizing binding of the colorant tocertain substrates. The use (or non-use) of a mordant in varioussituations can be dependent on the particular substrate, i.e., materialto be dyed (e.g., cellulosic fibers are more likely to require a mordantfor effective binding) and the conditions employed in the dyeingprocess.

The substrate can be in any form, e.g., in the form of fibers, woven ornonwoven textiles, fabrics, and the like. Substrate materials that canbe effectively dyed using the colorants described herein are notparticularly limited. Such substrates may be natural or synthetic, orcan comprise a combination of natural and synthetic components.Exemplary substrates include, but are not limited to, those comprisingprotein fibers (e.g., wool or silk) and cellulosic fibers (e.g.,cotton). In other embodiments, substrates, e.g., textiles derived fromtobacco plant fibers, e.g., as disclosed in U.S. Pat. No. 8,690,966 andU.S. Patent Application Publication Nos. 2015/0000050; 2012/0272460; and2011/0287681, all to Devall (which are incorporated herein by reference)can be dyed using the methods and/or colorants disclosed herein.Substrates can be employed in unprocessed form or can be processed priorto contact with the colorants disclosed herein. For example, thesubstrates can optionally be pre-pre-scoured (e.g., with a non-ionicwetting agent and/or scouring agent, such as a pectate lyase) and/or canbe pre-treated, e.g., with an alkaline material (e.g., via alkalinecationic pretreatment).

In some embodiments, dyeing of substrates with the colorants disclosedherein above is particularly effective for substrates comprising proteinfibers (e.g., wool and silk) and may provide enhanced depth ofshade/saturation for such substrates. An enhanced depth ofshade/saturation may be achieved in the absence of mordants,particularly with respect to protein fiber-containing substrates.Although cellulosic-based substrates (e.g., cotton) can be effectivelydyed also without mordants, mordants can be particularly beneficial forsuch substrates to improve the exhaustion of the natural tobaccocolorants thereon. A range of mordants are known and/or currently indevelopment to provide this function, e.g., as described in Handbook ofNatural Colorants, ed. Thomas Bechtold Wiley 2009 978-0-470-51199-2,which is incorporated herein by reference in its entirety. Exemplary,non-limiting examples of mordants include metal salts.

In certain embodiments, a colorant can be obtained as disclosed hereinand optionally stored and/or processed for further use, followed byemploying it to dye a given substrate. As such, dyeing can generallycomprise contacting a substrate with the colorant 170, which can beemployed in various forms (e.g., concentrated form, diluted form,pH-adjusted form, etc.). For example, the method can, in one embodiment,comprise contacting a substrate with colorant 170 and heating theresulting mixture (e.g., to at least about 40° C., such as between about40° C. and about 80° C., e.g., at about 60° C.). Following this heatingstep, various rinsing and draining steps can be conducted using water ofvarious temperatures. Scouring agents and pre-treatment agents can beadded before or after contacting the substrate with colorant 170. The pHof the substrate-containing mixture can be adjusted as desired.

In some embodiments, it has been advantageously found that the methodsof colorant production described herein above can be coupled with dyeingprocesses, such that the two processes can be conducted in situ. In suchembodiments, for example, natural colorants are extracted from a tobaccomaterial and are applied to one or more substrates within the same bath.Such methods are particularly applicable for the coloration of naturalsubstrates (e.g., natural fiber-containing materials), although they arenot limited thereto. Combining the extraction (to obtain the colorant)and dyeing processes into a common bath has been demonstrated to lead toa wide range of naturally colored substrates using less resources andtime. These methods can, in certain embodiments, employ the samereagents as disclosed herein above; however, these regents are employed,e.g., in different combinations and/or in the presence of the substrateto be dyed.

As such, in certain embodiments, tobacco-derived colorants can beobtained and immediately transferred to substrates. In such in situmethods, a substrate is added directly to the colorant-containing brothfollowing production. For example, the substrate can be added to thebroth immediately following step 22 as represented in FIGS. 1 and 2. Assuch, in one embodiment, the substrate can be added when colorant 170 isat elevated temperature (from treating at an elevated temperature duringstep 22). Although the colorant 170 can first be cooled from elevatedtemperature before contacting the substrate to be dyed with thecolorant, it can be advantageously in some embodiments to simplymaintain the colorant-containing broth at elevated temperature for thedyeing process. In some embodiments, the substrate is added to thecolorant-containing broth at elevated temperature and held at elevatedtemperature for a period of time, which is not particularly limited(e.g., at least about 10 minutes, at least about 20 minutes, at leastabout 30 minutes, at least about 60 minutes, etc.), but which issufficient to allow for at least some transfer of color from the brothto the substrate. The dyed material can then be removed, rinsed, anddried to give a dyed substrate.

In another embodiment, the substrate to be dyed can be added at anearlier stage of the colorant production process. Other non-limitingsteps at which the substrate can be added include during or afterproduction of Tobacco Mixture 1 130, during treatment step 20, followingthe first enzyme treatment (by adding to Treated Tobacco Mixture 1 140),during or after production of Tobacco Mixture 2 160, or during treatmentstep 22. In one particular embodiment, the substrate to be dyed is addeddirectly as a component of Tobacco Mixture 1 130 and thus is presentthroughout all enzyme treatment steps. It is noted that, in suchembodiments, it may be beneficial to incorporate one or more additionalcomponents in any of the mixtures disclosed herein, e.g., to facilitatethe dyeing. For example, in one embodiment, a pretreatment agent (e.g.,an alkaline cationic pretreatment agent) is incorporated along with thesubstrate to be dyed. The in situ method can, in some embodiments, beadvantageous in extracting color from the tobacco material andtransferring such color to the substrate to be dyed with decreasedoxidation prior to the transfer, e.g., without significant oxidation.

Dyed materials colored using the tobacco-derived colorants disclosedherein are generally permanently dyed or substantially permanently dyed.The dyed materials can, in some embodiments, be highly resistant tofading. The dyed materials advantageously have low content of variouscompounds initially associated with the tobacco material. For example,in preferred embodiments, the dyed materials contain little to nonicotine, e.g., less than about 0.1% by weight, less than about 0.09% byweight, or less than about 0.08 wt. %. The content of alkaloids otherthan nicotine is also advantageously low. For example, dyed materialsgenerally comprise little to no tobacco-specific nitrosamines (TSNAs).Exemplary TSNA compounds include N-nitrosonornicotine (NNN),4-methyl-N-nitrosamino-1-(3-pyridyl)-1-butanone (NNK),N-nitrosoanatabine (NAT), 4-methyl-N-nitrosamino-1-(3-pyridyl)-1-butanol(NNAL), and N-nitrosoanabasine (NAB). For example, in some embodiments,dyed materials prepared using the tobacco-derived colorant brothsdisclosed herein can contain less than about 0.02% by dry weightalkaloids other than nicotine. In certain embodiments, dyed substratesas disclosed herein uniquely exhibit a particular color independent oflighting. Typically, substrates treated with dyes and colorants exhibitdifferent shades; however, in some embodiments, substrates treated withthe tobacco-derived colorants disclosed herein exhibit color that isindependent of the lighting in which they are viewed.

EXPERIMENTAL

The present invention is more fully illustrated by the followingexample, which is set forth to illustrate the present invention and isnot to be construed as limiting thereof. Unless otherwise noted, allparts and percentages are by weight, and all weight percentages areexpressed on a dry basis, meaning excluding water content, unlessotherwise indicated.

Example 1: Natural Colorants Extracted from Tobacco (Plant Materials,Manufacturing by-Products, and/or Production Waste Streams) UsingAdvanced Enzymatic Techniques Enzymatic Extraction—Full Kettle Sequence(K Scrap Medium Grind) (Production Procedure for 20 Gallons of RawBroth)

Water (63,560 mL) is added to a 30 gallon steam jacket kettle. To thewater is added 10 g/l monosodium phosphate (MSP) (720 grams) to adjustthe pH to 5.5-6.0). Primafast Gold HSL (cellulase), 33% owg (2400 g),Multifect CX 15L (cellulase, beta-glucosidase), 16.5 owg (1200 g) and KScrap Medium (7264 g) are added to the pH-adjusted water. The resultingmixture is heated to 150° F. (65.6° C.) and held at this temperature for4-6 hours with constant agitation. Disodium phosphate (DSP) in an amountof 20 g/L (1440 grams) to adjust the pH to 7.5-8.0, 16.5% owg PRIMAGREENEcoScour (pectate lyase) (1200 grams), 33% owg Optimase CX 72L(xylanase) (2400 g), and water (9080 mL) are added. The temperature ofthis mixture is maintained at 150° F. (65.6° C.) for 6-8 hours withconstant agitation. The temperature of the mixture is then increased to185° F. (85° C.) and held at that temperature for 30 minutes withconstant agitation, followed by cooling overnight via radiation. Theresulting cooled mixture is filtered through a coarse strainer andstored in the refrigerator.

This general procedure was repeated for various tobacco materials toprovide various colorant-containing broths. Each such broth wasfreeze-dried and subjected to analysis for various sugars (by LC/MSMS)and alkaloids (by GC/MS), with the results presented below in Tables 1and 2. The various tobacco types referenced in the Tables below are asfollows: “Dark-cured” is dark air cured tobacco or dark fire-curedtobacco; “Scrap A” is tobacco in finely cut/dust form after tobaccoprocessing; “Processing waste” is a decanter pellet of tobacco materialproduced during protein purification, as disclosed, e.g., in U.S. Pat.No. 9,301,544, which is incorporated herein by reference in itsentirety; “Low-nicotine” is a variety of tobacco; “Red Russian” is avariety of tobacco; “Scrap B” is tobacco in finely cut/dust form aftertobacco processing from a different processing plant (which comprises amixture of tobacco types).

TABLE 1 Analysis of Sugars in Colorant-Containing Broths Reducing TotalSpecific Sugars (%) Sample Sugar (%) Sugar (%) Fructose Glucose SucroseDark fire cured 5.37 5.47 0.87 1.55 <0.16 Dark fire cured 6.92 6.91 1.081.15 <0.16 in situ* Scrap A 3.43 3.39 <0.12 1.49 <0.16 Scrap A in situ3.14 2.95 <0.12 0.75 <0.16 Processing waste 11.5 11.1 0.32 7.88 <0.16Processing waste 5.9 5.46 0.37 1.35 0.19 in situ* Low Nicotine 10.1 10.12.64 3.99 <0.16 variety Low Nicotine 11.6 11.8 3.49 3.28 <0.16 varietyin situ* Red Russian 16.8 16.6 4.45 8.92 <0.16 Red Russian in 20.4 20.66.52 9.94 0.4 situ* Scrap B 12.4 11.9 <0.12 8.12 <0.16 Scrap B in situ*7.27 6.97 <0.12 2.1 <0.16 *In situ samples are prepared by obtaining acolorant from the designated tobacco material and using the colorant dyeto dye the designated material in the same bath.

TABLE 2 Analysis of TSNAs in Colorant-Containing Broths Specific TSNAsAlkaloid NAB NAT NNK NNN Sample (%) (ng/g) (ng/g) (ng/g) (ng/g) Darkfire cured 0.638 13.7 358 152 463 Dark fire cured 0.846 19.9 506 174 646in situ* Scrap A 1.74 120 2580 1250 4240 Scrap A in situ* 2.39 174 33901420 5600 Processing waste 0.336 <9.77 40.7 <40.3 <40.7 Processing waste0.422 <10.0 77.5 <41.2 <41.6 in situ* Low Nicotine 0.165 <10.0 <40.2<41.4 <41.8 variety Low Nicotine 0.158 <10.0 <40.1 <41.3 <41.7 varietyin situ* Red Russian <0.100 <9.93 <39.8 <40.9 <41.4 Red Russian in 0.138<9.97 <40.0 <41.1 <41.5 situ* Scrap B <0.100 <10.4 43.4 212 <43.4 ScrapB in situ* 0.148 <10.3 51.9 157 51.9 *In situ samples are prepared byobtaining a colorant from the designated tobacco material and using thecolorant dye to dye the designated material in the same bath.

Example 2: Dyeing of Natural Fibers with Natural Colorants Extractedfrom Tobacco (Plant Materials, Manufacturing by-Products, and/orProduction Waste Streams) Using Combination of Natural and TextileChemistry

Dyeing Procedure—Using Color Extracted from Tobacco

The fabric dyed is 1500 grams, 100% cotton knit greige. The fabric isfirst treated in a Optidye Sample Dye machine (rated 5 pound maximumcapacity), which is a garment dye extractor. The fabric is prepared bypre-scouring by the following process. First, the fabric is loaded intoa dye machine (approximately 1000 g) and the machine is filled to 20liters using cold water. Non-ionic wetting agent (Rucowet FINConcentrate, from Rudolf) (3 g) is added and PrimaGreen EcoScour(pectate lyase from Dupont™ Enzymes) (30 g) is added. The mixture isheated to 65° C. and held at that temperature for 30 minutes, afterwhich time the machine is drained and filled to 20 liters using coldwater. The mixture is rinsed for 3 minutes, drained, and the water isremoved using an extraction spin cycle (250 rpm/2 minutes).

The fabric is next subjected to cationic polymer pretreatment. Themachine is filled to 15 liters using cold water and PT92L (Organic Chem.cationic polymer) (10% owg, 150 g) is slowly added over 6 minutes. Theresulting mixture is held for 10 minutes, followed by the addition of 5%owg soda ash (75 g) slowly over 4 minutes. The mixture is heated to 60°C. and held at that temperature for 60 minutes, after which time themachine is drained and filled to 20 liters using cold water. Optionally,acetic acid 56% (40 g) is added. The material is rinsed for 5 minutesand drained with no extraction.

The fabric is then treated with natural colorant coupled with cationicpolymer. The dye machine is filled to 15 liters using cold water and themachine is run/agitated for 5 minutes. Tobacco-derivedcolorant-containing broth (as prepared according to Example 1, above) isadded slowly over 6 minutes. The mixture is heated to 60° C. and held atthat temperature for 60 minutes, after which time the machine is drainedand filled to 20 liters using cold water. The material is rinsed for 3minutes, the machine is drained, and filled to 20 liters using hotwater. Non-ionic scouring agent (5 g) is added to the mixture and themixture is heated to 85° C. and soaked at that temperature for 3minutes, after which time the machine is drained and filled to 20 litersusing tempered water. The material is rinsed for 3 minutes, drained, andthe machine is filled to 15 liters using cold water. The material isrinsed for 3 minutes, after which time the machine is drained and thematerial extracted (250 rpm/2 minutes). The colored fabric is tumbledried for 45 minutes at 70° C.

Example 3: In Situ Extraction of Natural Colorants Extracted fromTobacco (Plant Materials, Manufacturing by-Products, and/or ProductionWaste Streams) Coupled with Application to Natural Fibers in the SameBath In Situ Extraction and Dyeing Process A:

In a 500 mL Erlenmeyer flask, tobacco raw material (20 g), GC(cellulase, beta gluconase) (10 g), 72L (xylanase) (10 g), DSP to pH 8,and water to a total volume of 200 mL are combined. The mixture isheated solely with constant agitation to 60° C. and held at thattemperature for 60-120 minutes. Alkaline cationic pre-treated cotton(pre-wet and squeezed) is added (10 g) and the mixture is held at 60° C.for 30-60 minutes. The dyed cotton is removed and rinsed (cold water/hotwater/cold water). A substantial portion of the water is removed fromthe dyed cotton by an extraction/spin cycle and the cotton is tumbledried.

In Situ Extraction and Dyeing Process B:

In a 500 mL Erlenmeyer flask, greige cotton knit (10 g), PT92L alkalinecationic pretreatment (0.6 g), tobacco raw material (10 g), GC(cellulase, beta gluconase) (5 g), 72L (xylanase) (5 g), DSP to pH 8.5,and water to a total volume of 200 mL are combined. The mixture isheated solely with constant agitation to 60° C. and held at thattemperature for 60-120 minutes. The dyed cotton is removed and rinsed(cold water/hot water/cold water). A substantial portion of the water isremoved from the dyed cotton by an extraction/spin cycle and the cottonis tumble dried.

Colored fabric samples prepared according to Examples 2 and 3 aresubjected to analysis by GC/MS to determine content of anabasine,anatabine, myosmine, nicotine, nornicotine, tobacco-specificnitrosamines (NAB, NAT, NNK, NNN) and total alkaloids. The results ofthe testing are provided in Tables 3 and 4, below.

TABLE 3 Analysis of Specific Alkaloids in Colored Fabrics AnabasineAnatabine Myosmine Nicotine Nornicotine Sample (%) (%) (%) (%)mg/cig^(#) (%) Cotton: control <0.0009 <0.0009 <0.0009 <0.0775 <0.77<0.0096 Cotton: scrap B <0.0009 <0.0009 <0.0009 <0.0779 <0.78 <0.0097Cotton: scrap A <0.0009 <0.0009 <0.0009 <0.0779 <0.78 <0.0097 Cotton:processing waste <0.0009 <0.0009 <0.0009 <0.0780 <0.78 <0.0097 Cotton:dark fire cured <0.0009 <0.0009 <0.0009 <0.0778 <0.78 <0.0097 Cotton: insitu* scrap B <0.0009 <0.0009 <0.0009 <0.0771 <0.77 <0.0096 Cotton:in-situ scrap A 0.0018 0.0009 0.009 <0.0780 <0.78 0.0097 Cotton: insitu* processing <0.0009 <0.0009 <0.0009 <0.0776 <0.78 <0.0096 wasteCotton: in situ* dark fire <0.0009 <0.0009 <0.0009 <0.0782 <0.78 <0.0097cured Cotton: in situ* low <0.0009 <0.0009 <0.0009 <0.0780 <0.78 <0.0097nicotine Cotton: in situ* Red <0.0009 <0.0009 <0.0009 <0.0777 <0.78<0.0097 Russian Cotton: low nicotine <0.0009 <0.0009 <0.0009 <0.0781<0.78 <0.0097 Cotton: Red Russian <0.0009 <0.0009 <0.0009 <0.0778 <0.78<0.0097 Wool: control No results (contaminated sample) Wool: scrap B<0.0009 0.0074 <0.0009 <0.0778 <0.78 <0.0097 Wool: in situ* scrap B<0.0009 <0.0009 <0.0009 <0.0779 <0.78 <0.0097 Wool: scrap A <0.0009<0.0009 <0.0009 <0.0780 <0.78 <0.0097 Wool: processing waste <0.0009<0.0009 <0.0009 <0.0781 <0.78 <0.0097 Wool: dark cure <0.0009 <0.0009<0.0009 <0.0769 <0.77 <0.0097 Wool: in-situ* scrap A <0.0009 0.0041<0.0009 <0.0778 <0.78 0.0097 Wool: in situ* processing <0.0009 <0.0009<0.0009 <0.0774 <0.77 <0.0096 waste Wool: in situ* dark fire <0.0009<0.0009 <0.0009 <0.0779 <0.78 <0.0097 cured Wool: in situ* low nicotine<0.0009 <0.0009 <0.0009 <0.0772 <0.77 <0.0096 Wool: in situ* Red Russian<0.0009 <0.0009 <0.0009 <0.0773 <0.77 <0.0096 Wool: Low nicotine <0.0009<0.0009 <0.0009 <0.0773 <0.77 <0.0096 Wool: Red Russian <0.0009 <0.0009<0.0009 <0.0780 <0.78 <0.0097 *In situ samples are prepared by obtaininga colorant from the designated tobacco material and using the colorantto dye the designated material in the same bath. ^(#)Mg/cig units areroughly equivalent to mg/g

TABLE 4 Analysis of Specific TSNAs and Total Alkaloids in ColoredFabrics TSNA Content (ng/g) Total Alkaloids (%) Sample NAB NAT NNK NNNwith nicotine without nicotine Cotton: control <7.51 <30.1 <31.0 <31.3<0.0898 <0.0123 Cotton: scrap B <7.58 <30.4 <31.3 <31.6 <0.0903 <0.0124Cotton: scrap A <7.67 <30.7 <31.6 <31.9 <0.0903 <0.0124 Cotton:processing waste <7.16 <28.7 <29.5 <29.8 <0.0904 <0.0124 Cotton: darkfire cured <7.09 <28.4 <29.2 <29.5 <0.0902 <0.0124 Cotton: in situ*scrap B <7.70 <30.9 <31.8 <32.1 <0.0894 <0.0123 Cotton: in-situ* scrap A<7.78 <31.2 <32.1 <32.4 <0.0913 0.0133 Cotton: in situ* processing <7.48<30.0 <30.8 <31.1 <0.0899 <0.0123 waste Cotton: in situ* dark fire <7.56<30.3 <31.2 <31.5 <0.0906 <0.0124 cured Cotton: in situ* low <7.51 <30.1<30.9 <31.3 <0.0904 <0.0124 nicotine Cotton: in situ* Red <7.97 <32.0<32.9 <33.2 <0.0901 <0.0124 Russian Cotton: low nicotine <7.50 <30.1<30.9 <31.2 <0.0905 <0.0124 Cotton: Red Russian <7.40 <29.6 <30.5 <30.8<0.0902 <0.0124 Wool: control No results (contaminated sample) Wool:scrap B <7.56 <30.3 <31.2 <31.5 <0.0967 <0.0189 Wool: in situ* scrap B<7.35 <29.5 <30.3 <30.6 <0.0903 <0.0124 Wool: scrap A <7.60 <30.5 <31.3<31.7 <0.0904 <0.0124 Wool: processing waste <7.54 <30.2 <31.2 <31.4<0.0905 <0.0124 Wool: dark fire cured <7.19 <28.8 <29.6 <29.9 <0.0892<0.0123 Wool: in-situ* scrap A <7.57 <30.4 <31.2 <31.5 <0.0934 <0.0156Wool: in situ* processing <8.03 <32.2 <33.1 <33.4 <0.0897 <0.0123 wasteWool: in situ* dark fire <7.15 <28.7 <29.5 <29.8 <0.0903 <0.0124 curedWool: low nicotine <7.27 <29.1 <30.0 <30.3 <0.0896 <0.0123 Wool: RedRussian <7.47 <29.9 <30.8 <31.1 <0.0904 <0.0124 Wool: in situ* lownicotine <7.91 <3.17 <32.6 <32.9 <0.0895 <0.0123 Wool: in situ* RedRussian <7.42 <29.7 <30.6 <30.9 <0.0896 <0.0123 *In situ samples areprepared by obtaining a colorant from the designated tobacco materialand using the colorant to dye the designated material in the same bath

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

1.-22. (canceled)
 23. A colored substrate prepared according to a methodcomprising: providing a first tobacco mixture by combining the tobaccomaterial with a cellulase and a beta glucosidase at a pH of about 5-6;providing a second tobacco mixture by adjusting the pH of the firsttobacco mixture to a pH of about 7-8 and adding pectate lyase andxylanase to the first tobacco mixture; separating the tobacco materialfrom the second tobacco mixture to give a colorant-containing broth; andcontacting a substrate with the colorant-containing broth to give thecolored substrate, wherein the colored substrate exhibits color that isindependent of lighting.
 24. The colored substrate of claim 23, whereinthe colored substrate is a dyed substrate.
 25. The colored substrate ofclaim 24, wherein the dyed substrate is permanently dyed.
 26. Thecolored substrate of claim 23, wherein the first tobacco mixturecomprises tobacco derived from a Nicotiana species selected from thegroup consisting of N. tabacum, N. rustica, N. alata, N. arentsii, N.excelsior, N. forgetiana, N. glauca, N. glutinosa, N. gossei, N.kawakamii, N. knightiana, N. langsdorffi, N. otophora, N. setchelli, N.sylvestris, N. tomentosa, N. tomentosiformis, N. undulata, N. xsanderae, N. africana, N. amplexicaulis, N. benavidesii, N. bonariensis,N. debneyi, N. longiflora, N. maritina, N. megalosiphon, N.occidentalis, N. paniculata, N. plumbaginifolia, N. raimondii, N.rosulata, N. simulans, N. stocktonii, N. suaveolens, N. umbratica, N.velutina, N. wigandioides, N. acaulis, N. acuminata, N. attenuata, N.benthamiana, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa,N. fragrans, N. goodspeedii, N. linearis, N. miersii, N. nudicaulis, N.obtusifolia, N. occidentalis subsp. Hersperis, N. pauciflora, N.petunioides, N. quadrivalvis, N. repanda, N. rotundifolia, N.solanifolia, N. spegazzinii, and combinations thereof.
 27. The coloredsubstrate of claim 23, wherein the first tobacco mixture comprisestobacco selected from the group consisting of flue-cured tobacco,Virginia tobacco, burley tobacco, sun-cured tobacco, Maryland tobacco,dark tobacco, dark-fired tobacco, dark air cured tobacco, light aircured tobacco, Indian air cured tobacco, Red Russian tobacco, Rusticatobacco, and combination thereof.
 28. The colored substrate of claim 23,wherein the first tobacco mixture comprises low-nicotine tobacco or RedRussian tobacco.
 29. The colored substrate of claim 23, wherein thefirst tobacco mixture comprises two or more tobacco types.
 30. Thecolored substrate of claim 23, wherein the first tobacco mixturecomprises tobacco that has been cured.
 31. The colored substrate ofclaim 30, wherein the tobacco comprises dark air-cured tobacco orfire-cured tobacco.
 32. The colored substrate of claim 23, wherein thefirst tobacco mixture comprises tobacco that has been subjected to aprotein extraction process.
 33. The colored substrate of claim 23,wherein the first tobacco mixture comprises tobacco in fine cut or dustform.
 34. The colored substrate of claim 23, wherein the first tobaccomixture comprises tobacco in cut leaf form.
 35. The colored substrate ofclaim 23, wherein the substrate comprises a natural material.
 36. Thecolored substrate of claim 23, wherein the substrate comprises asynthetic material.
 37. The colored substrate of claim 23, wherein thesubstrate is a paper, plastic, ceramic, or metal.
 38. The coloredsubstrate of claim 23, wherein the substrate is a woven or non-woventextile.
 39. The colored substrate of claim 23, wherein the substratecomprises protein fibers.
 40. The colored substrate of claim 39, whereinthe colorant-containing broth does not include a mordant.
 41. Thecolored substrate of claim 23, wherein the substrate comprises fibersselected from the group consisting of wool fibers, silk fibers, cottonfibers, and combinations thereof.
 42. The colored substrate of claim 23,wherein the substrate comprises a textile derived from tobacco plantfibers.
 43. The colored substrate of claim 23, wherein the coloredsubstrate comprises less than about 0.1% by weight of nicotine.